Kiyoshi Ito

Crystal Structure of Aminopeptidase N (Proteobacteria Alanyl Aminopeptidase) from Escherichia coli and Conformational Change of Methionine 260 Involved in Substrate Recognition

Aminopeptidase N from Escherichia coli is a broad specificity zinc exopeptidase belonging to aminopeptidase clan MA, family M1. The structures of the ligand-free form and the enzyme-bestatin complex were determined at 1.5- and 1.6-Å resolution, respectively. The enzyme is composed of four domains: an N-terminal β-domain (Met1-Asp193), a catalytic domain (Phe194-Gly444), a middle β-domain (Thr445-Trp546), and a C-terminal α-domain (Ser547-Ala870). The structure of the catalytic domain exhibits similarity to thermolysin, and a metal-binding motif (HEXXHX18E) is found in the domain. The zinc ion is coordinated by His297, His301, Glu320, and a water molecule. The groove on the catalytic domain that contains the active site is covered by the C-terminal α-domain, and a large cavity is formed inside the protein. However, there exists a small hole at the center of the C-terminal α-domain. The N terminus of bestatin is recognized by Glu121 and Glu264, which are located in the N-terminal and catalytic domains, respectively. Glu298 and Tyr381, located near the zinc ion, are considered to be involved in peptide cleavage. A difference revealed between the ligand-free form and the enzyme-bestatin complex indicated that Met260 functions as a cushion to accept substrates with different N-terminal residue sizes, resulting in the broad substrate specificity of this enzyme.

Importance of arginine at position 170 of the A subunit of Vero toxin 1 produced by enterohemorrhagic Escherichia coli for toxin activity

Comparison of the primary structures of the A subunits of Vero toxin 1 (VT1), Vero toxin 2 (VT2), and two variants of VT2 (VT2vp and VT2vh) and the ricin A chain revealed three conserved regions (amino acid residues 51-55, 167-171 and 202-207 from the N-terminus of VT1). All three regions of the ricin A chain corresponded in position to the active site of ricin proposed by X-ray crystal diffraction analysis. To determine the relative importance of the conserved amino acid residues for toxin activity of VT1, we prepared VT1 mutants with single amino-acid substitutions by oligonucleotide-directed site-specific mutagenesis. A total of 22 mutants were prepared to examine 14 conserved residues, and their cytotoxicities to Vero cells and inhibitory activities on protein synthesis in a rabbit reticulocyte lysate were compared with those of wild-type VT1. Replacement of glutamic acid at position 167 by glutamine and of arginine at position 170 by leucine reduced both activities drastically. These results suggest that, in addition to the glutamic acid at position 167 reported previously, arginine at position 170 also plays an important role in the toxin activity of VT1. A possible chemical mechanism of the enzymatic (N-glycosidase) activity of VT1 is proposed based on the relative activities of various mutants.

Inhibition of protein synthesis by a Vero toxin (VT2 or Shiga-like toxin II) produced by Escherichia coli O157 : H7 at the level of elongation factor 1-dependent aminoacyl-tRNA binding to ribosomes

A Vero toxin (VT2 or Shiga-like toxin II) from Escherichia coli O157:H7 was shown to inhibit protein synthesis in a rabbit reticulocyte lysate, but not in wheat germ or Ercherichia coli lysates. The toxin, VT2, inactivated 60S ribosomal subunits of rabbit reticulocytes. The site of inhibition of protein synthesis by VT2 was shown to be elongation factor 1-dependent aminoacyl-tRNA binding to ribosomes. VT2 did not affect Met-tRNAf binding to ribosomes, non-enzymatic binding of aminoacyl-tRNA to ribosomes, peptide bond formation or translocation.

Relationship among activation of the Na+/H+ antiporter, ornithine decarboxylase induction, and DNA synthesis

The relationship among activation of the Na+/H+ antiporter, ornithine decarboxylase, and DNA synthesis was examined with bovine small lymphocytes stimulated by concanavalin A (Con A). The Na+/H+ antiport activity was activated immediately after addition of concanavalin A; the maximum was reached 1 h after Con A addition and the activation continued at least 6 h. With increasing concanavalin A concentrations, the activities of the Na+/H+ antiporter, ornithine decarboxylase, and DNA synthesis increased in a parallel manner. In the presence of HCO3- in the medium, the internal alkalinization of lymphocytes was not induced by Con A. Ornithine decarboxylase and DNA synthetic activities were not inhibited by 5-(N-ethyl-N-isopropyl) amiloride (EIPA), a specific inhibitor of the Na+/H+ antiporter. In contrast, in the absence of HCO3- in the medium, the internal pH was alkalinized approximately 0.06 pH units by Con A. EIPA did inhibit the alkalinization of the internal pH or DNA synthesis significantly. Ornithine decarboxylase activity was not inhibited by EIPA. These results indicate that the activation of a Na+/H+ antiporter is not a trigger for cell proliferation, but its activation is important probably through the maintenance of the internal pH optimum, especially in HCO3(-)-free medium.

Comparison of the modes of action of a vero toxin (a Shiga-like toxin) from Escherichia coli, of ricin, and of α-sarcin

The modes of action of a Vero toxin (VT2 or Shiga-like toxin II) from Escherichia coli, of ricin, and of alpha-sarcin were compared. Elongation factor 1 (EF1) and GTP-dependent Phe-tRNA binding to ribosomes in the presence of poly(U) was inhibited by these three toxins, but EF1 and guanylyl (beta, gamma-methylene)-diphosphate-dependent Phe-tRNA binding was inhibited by alpha-sarcin only. EF1- and Phe-tRNA-dependent GTPase activity was inhibited by these toxins, but nonenzymatic binding of Phe-tRNA was not. The turnover rate of EF1 binding to ribosomes during Phe-tRNA binding was also decreased by these three toxins. The addition of EF1 recovered the inhibition of Phe-tRNA binding to ribosomes by VT2 and ricin but not by alpha-sarcin. The formation of and EF2- and GTP-dependent puromycin derivative of phenylalanine was inhibited slightly by the three toxins, indicating that translocation is not influenced significantly by them. EF2-dependent GTPase activity was stimulated by these toxins, and especially by VT2 and ricin. In contrast, the binding of EF2 to ribosomes was inhibited strongly by VT2 and ricin, and slightly by alpha-sarcin. The stimulation of EF2-dependent GTPase activity by the toxins may compensate for the decrease of EF2 binding to ribosomes which they caused during translocation. In total, these results indicate that VT2 and ricin inhibit protein synthesis through the disturbance of the turnover of EF1 binding to ribosomes during aminoacyl-tRNA binding to ribosomes, and that alpha-sarcin inhibits the synthesis through the inhibition of the binding of the complex of Phe-tRNA, EF1, and GTP to ribosomes.

Structure of aminopeptidase N from Escherichia coli complexed with the transition-state analogue aminophosphinic inhibitor PL250

Aminopeptidase N (APN; EC 3.4.11.2) purified from Escherichia coli has been crystallized with the optically pure aminophosphinic inhibitor PL250, H3N+‐CH(CH3)‐P(O)(OH)‐CH2‐CH(CH2Ph)‐CONH‐CH(CH2Ph)CO2 −, which mimics the transition state of the hydrolysis reaction. PL250 inhibits APN with a K i of 1.5–2.2 nM and its three‐dimensional structure in complex with E. coli APN showed its interaction with the S1, S′1 and S′2 subsites of the catalytic site. In this structure, the Zn ion was shown to be pentacoordinated by His297, His301 and Glu320 of APN and the two O atoms of the phosphinic moiety of PL250. One of these O atoms is also involved in a hydrogen bond to Tyr381, supporting the proposed role of this amino acid in the stabilization of the transition state of the enzymatic process. The strength of the phosphinic zinc binding and the occupancy of the S′2 subsite account for the 100‐fold increase in affinity of PL250 compared with the dipeptide‐derived inhibitor bestatin (K i = 4.1 × 10−6  M). Accordingly, the removal of the C‐terminal phenylalanine of PL250 resulted in a large decrease in affinity (K i = 2.17 × 10−7  M). Furthermore, it was observed that the C‐terminal carboxyl group of the inhibitor makes no direct interactions with the amino acids of the APN active site. Interestingly, PL250 exhibits the same inhibitory potency for E. coli APN and for mammalian enzymes, suggesting that the structure of the complex could be used as a template for the rational design of various human APN inhibitors needed to study the role of this aminopeptidase in various pathologies.

Demonstration of RNA N -Glycosidase Activity of a Vero Toxin (VT2 Variant) Produced by Escherichia coli O91:H21 from a Patient with the Hemolytic Uremic Syndrome

A new Vero toxin purified from Escherichia coli O91: H21 isolated from a patient with the hemolytic uremic syndrome (VT2vh) was shown to inhibit elongation factor 1‐dependent aminoacyl‐tRNA binding to ribosomes, resulting in inhibition of protein synthesis in rabbit reticulocytes. VT2vh, like Shiga toxin, VT1 and VT2, showed RNA N‐glycosidase activity and cleaved the N‐glycosidic bond of the adenosine residue at position 4324 in 28S ribosomal RNA.

Polyamine regulation of the synthesis of thymidine kinase in bovine lymphocytes.

Concanavalin A-activated lymphocytes were made polyamine deficient by treatment with alpha-difluoromethylornithine and ethylglyoxal bis(guanylhydrazone). Thymidine kinase activity in polyamine-deficient cells was 17% of the level in normal cells. Thymidine kinase mRNA increased with time after concanavalin A activation and reached a maximum at 36 h after concanavalin A addition. The amount of thymidine kinase mRNA in polyamine-deficient cells was approximately 75% of that in normal cells. The transcription of thymidine kinase gene in isolated nuclei of polyamine-deficient cells was also 75% of that from normal cells. The turnover rate of thymidine kinase mRNA in both normal and polyamine-deficient cells was nearly equal. In normal cells, 95% of thymidine kinase mRNA was polysome associated, while in polyamine-deficient cells, 60% of the mRNA was polysome associated. In addition, the size of polysomes associated with thymidine kinase mRNA in polyamine-deficient cells was smaller than that in normal cells. Synthesis of thymidine kinase was stimulated approximately seven-fold by 0.3 mM spermidine in a rabbit reticulocyte polyamine-free protein synthetic system. The half-life of thymidine kinase activity in both normal and polyamine-deficient cells was nearly equal. Thymidine kinase activity was not influenced significantly by 0.3 mM spermidine. These combined results suggested that the synthesis of thymidine kinase was mainly regulated by polyamines at the level of translation.

Characterization of glucoamylase and α-amylase from Monascus anka: Enhanced production of α-amylase in red koji

To enhance glucoamylase and α-amylase production from Monascus anka, we investigated the influence of different culture conditions on enzyme production and purified and characterized these enzymes. The effect of different raw materials was investigated by using solid-state plates of raw materials such as barley and non-waxy or waxy rice. The barley plate was the most suitable for mycelial growth, but glucoamylase and α-amylase production per growth area did not differ according to the raw material. Investigation of the effect of temperature showed that incubation at 37 °C promoted maximal cell growth, while incubation at 25 °C and at 40 °C resulted in enhanced α-amylase and glucoamylase production, respectively. Characterization of the purified enzymes revealed that α-amylase was unstable at acidic pH and less resistant to heat (stable at < 40 °C) than glucoamylase. When these culture conditions were applied to enzyme production in red koji, reducing the temperature from 35 °C to 25 °C for 48 h in the late stages of growth resulted in higher glucoamylase and α-amylase production (1.4 and 18 times, respectively) with a concomitant increase in protein stability.

Dipeptidyl Aminopeptidase IV from Stenotrophomonas maltophilia Exhibits Activity against a Substrate Containing a 4-Hydroxyproline Residue

The crystal structure of dipeptidyl aminopeptidase IV from Stenotrophomonas maltophilia was determined at 2.8-A resolution by the multiple isomorphous replacement method, using platinum and selenomethionine derivatives. The crystals belong to space group P4(3)2(1)2, with unit cell parameters a = b = 105.9 A and c = 161.9 A. Dipeptidyl aminopeptidase IV is a homodimer, and the subunit structure is composed of two domains, namely, N-terminal beta-propeller and C-terminal catalytic domains. At the active site, a hydrophobic pocket to accommodate a proline residue of the substrate is conserved as well as those of mammalian enzymes. Stenotrophomonas dipeptidyl aminopeptidase IV exhibited activity toward a substrate containing a 4-hydroxyproline residue at the second position from the N terminus. In the Stenotrophomonas enzyme, one of the residues composing the hydrophobic pocket at the active site is changed to Asn611 from the corresponding residue of Tyr631 in the porcine enzyme, which showed very low activity against the substrate containing 4-hydroxyproline. The N611Y mutant enzyme was generated by site-directed mutagenesis. The activity of this mutant enzyme toward a substrate containing 4-hydroxyproline decreased to 30.6% of that of the wild-type enzyme. Accordingly, it was considered that Asn611 would be one of the major factors involved in the recognition of substrates containing 4-hydroxyproline.